Project Summary Adolescent girls in sub-Saharan Africa are disproportionately affected by sexually transmitted infections (STIs) and HIV. Sexually active African adolescents are known to have high levels of vaginal inflammation. We hypothesize that even pre-sexual debut, African adolescent girls may have elevated vaginal inflammation and elevated vaginal microbial diversity, which will influence their genital immunological maturation, microbiome, and later susceptibility to STIs. We further hypothesize that sexual debut may result in persistent genital inflammation and increased bacterial diversity. We will extend a pre-existing 3 year cohort of adolescent girls in Thika, Kenya, recruited between ages 16-18 and pre-sexual debut, and follow them 2 additional years, for a total of 5 years, and determine incident gonorrhea, chlamydia, and trichomonas infection. Our first aim is to conduct a case-control study comparing the inflammatory profiles in cervicovaginal lavage (measured by cytokines IL-6, IP-10, IL-1a and MIP-1b and others) of girls who acquire STIs with controls. We will also compare baseline density of inflammatory cells of vulvar biopsy specimens between cases and controls, and we will compare broad-range PCR measurements of vaginal microbial diversity, and quantification by PCR of specific high-risk anaerobes between girls who acquire STIs and controls. Our second aim is to analyze pre- and post-sexual debut changes in inflammation, microbial diversity, and cell density. We also will analyze the role of progestin-based contraceptives and lifetime estrogen exposure (time from menarche to sexual debut) to evaluate the effects of hormonal variation on STI risk factors. We will collaborate with expert immunologists to perform rigorous MSD analysis of vaginal inflammatory markers, use cutting edge histopathological methods to evaluate tissue inflammation, and propose innovative full-length amplicon microbiome measurement techniques to overcome imprecision in urogenital species identification. Our approach is efficient as it leverages and extends an existing adolescent cohort with high retention rates, and is unique as we have successfully collected pre-sexual debut cervicovaginal lavage, microbiota and vulvar biopsy specimens from adolescent girls. Our results will provide a comprehensive picture of pre-sexual debut immunology of African adolescent girls, identify factors that promote STI acquisition, and will further demonstrate evolution of girls' genital tract immunity and microbiome as they begin having sex. We expect that this approach will identify modifiable biological risk factors to reduce STI acquisition among adolescent girls, and results can be used to develop STI prevention methods and multi-purpose technologies.